[show abstract][hide abstract] ABSTRACT: In the human stomach, the peptide TFF2 is secreted together with the mucin MUC6 by mucous neck cells and antral gland cells. TFF2 is strongly associated with the gastric mucus and promotes gastric restitution. Here, TFF2 was purified from the human corpus and antrum, respectively, by size exclusion chromatography, and the N-linked glycan structure at N-15 of the mature peptide determined. As a hallmark, the unusual monofucosylated N,N'-diacetylhexosediamine (tentatively assigned as GalNAcβ1→4GlcNAc, LacdiNAc) modification was detected as the terminal structure of a bi-antennary complex type N-glycan exhibiting also core fucosylation. Replicate analyses did not show microheterogeneities in the fraction of PNGaseF cleaved and permethylated N-glycans when analyzed by MALDI-mass spectrometry. On the glycopeptide level a minor glycan microheterogeneity was evident in liquid chromatography-ESI-mass spectrometry demonstrating the presence of underfucosylated species. The tryptic TFF2 N-glycopeptide p34-39 (LSPHNR N-glycosylated with Fuc3Hex3HexNAc6) was identified both by ESI-MS/MS and MALDI-post-source decay analysis. Lectin analyses with the Wisteria floribunda agglutinin indicated the potential presence of LacdiNAc terminating glycans and revealed minor differences between TFF2 from fundic units, i. e., mucous neck cells, and antral units, i. e. antral gland cells. Strikingly, on the level of the primary structure there was no indication that formation of the proposed LacdiNAc structure is cis-controled by a peptidic determinant related to published sequences.
[show abstract][hide abstract] ABSTRACT: The gastric mucosa is subject to continual bidirectional renewal by differentiation from stem and transit amplifying cells. It was the aim of this study to characterize the self-renewal of the human gastric mucosa and its two major types of glands in the fundus and antrum, respectively. Three characteristic regions (pit, proliferative, and lower neck regions) were isolated from fundic and antral units by the use of laser microdissection, and expression profiles concerning 15 marker genes were generated by RT-PCR analysis. The surface mucous cells (SMCs) of fundic and antral units differed in their expression of at least four secretory genes, i.e., gastric lipase, TFF3, FCGBP, and lysozyme. The maturation of mucous neck cells was shown to occur stepwise, first towards a mucous phenotype followed by a serous differentiation step. Also, a stepwise maturation of both the antral SMCs and antral gland cells was observed. Additionally, the presence of gastric lipase was also demonstrated for the first time in antral gland cells. In conclusion, the different expression profiles of SMCs of the fundic and antral units could be the basis for the different self-renewal rates of fundic and antral SMCs and could influence the spatial organization of the bacterial microbiota within the various parts of the gastric mucosa.
[show abstract][hide abstract] ABSTRACT: TFF3 is a secretory peptide belonging to the trefoil factor family with a predicted size of 59 amino acid residues containing seven cysteine residues. It is predominantly expressed in intestinal goblet cells where it plays a key role in mucosal regeneration and repair processes. In the course of these studies, human colonic TFF3 was shown to exist mainly as a high molecular weight heteromer. Purification of this heteromer and characterization by LC-ESI-MS/MS analysis identified the IgG Fc binding protein (FCGBP) as the disulfide-linked partner protein of TFF3. FCGBP is a constituent of intestinal mucus secreted by goblet cells. Furthermore, low amounts of TFF3/monomer and only little TFF3/dimer were detected in human colonic extracts. Here, we show that these TFF3 forms can be released from the purified TFF3-FCGBP heteromer complex in vitro by reduction with hydrogen sulfide (H(2)S). Such a mechanism would be in line with the high H(2)S concentrations reported to occur in the lumen of the colon. Of special note, this points to intestinal mucus as a reservoir for a biologically active peptide. Also proteolytic processing of FCGBP was observed which is in line with multiple autocatalytic cleavages as proposed earlier by Johansson et al. (J. Proteome Res. 2009 , 8 , 3549 - 3557).
Journal of Proteome Research 06/2010; 9(6):3108-17. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Trefoil factor family (TFF) peptides promote regeneration and repair processes of mucous epithelia. They also probably play a key role in the remarkable regenerative capacity of the urinary tract epithelia. We have localized TFF1, TFF2, and TFF3 expression systematically in surgical specimens from the urinary tract by reverse transcription with the polymerase chain reaction, Western blot analysis, and immunohistochemistry. Urine samples from patients suffering from nephrolithiasis have been investigated and compared with those of healthy controls. TFF synthesis is detectable along the entire urinary tract epithelia. TFF3 synthesis is the most pronounced followed by TFF1, whereas TFF2 synthesis is occasionally detectable but only in trace amounts. In contrast, TFF2 is the predominant TFF peptide excreted into the urine, and significantly increased urinary TFF2 levels (together with occasionally raised TFF3 levels) have been observed in patients suffering from nephrolithiasis. Thus, we consider that TFF3 plays a major part in regeneration and restitution processes in urinary tract epithelia. TFF2 and probably also TFF3 are candidate biomarkers for nephrolithiasis and possibly other inflammatory conditions of the urinary tract.
Cell and Tissue Research 03/2010; 339(3):639-47. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Both the major and minor salivary glands are the sources of saliva, a fluid vital for the maintenance of a healthy oral cavity. Here, the expression profiles of human submandibular (SMG) and labial glands (LG) were compared by RT-PCR analysis of laser microdissected mucous and serous cells, respectively. The focus was on trefoil factor family (TFF) genes, but also other genes encoding secretory proteins (mucins, lysozyme, amylase, statherin, and histatins) or aquaporin 5 were included. Immunofluorescence studies concerning TFF1-3, FCGBP, amylase, and lysozyme are also presented. It was shown that LGs clearly contain serous cells and that these cells differ in their expression profiles from serous SMG cells. Furthermore, all three TFF peptides, together with MUC5B, MUC7, MUC19, and FCGBP, were clearly detectable in mucous acini of both LGs and SMGs. In contrast, lysozyme was differentially expressed in LGs and SMGs. It can be expected that labial saliva may play a particularly important role for protecting the teeth.
Cellular Physiology and Biochemistry 01/2010; 26(3):375-82. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mucous epithelia represent a major barrier to the outside world and are capable of undergoing rapid repair after injury by cell migration, a process called "restitution". Here, a sensitive RT-PCR method was applied allowing systematic gene expression analysis of separated stationary and migratory non-transformed IEC-18 and IEC-6 cells after scratch wounding. The focus was on genes related to cell-cell contacts. Furthermore, the effect of epidermal growth factor (EGF) on gene expression was studied. Most of the genes investigated here were down-regulated in migratory cells. Many of the alterations are expected to affect the permeability of tight junctions. Also the nectin-afadin complex of adherens junctions was modulated as well as the expression of both the chemokine receptor CXCR4 and the EGF receptor. Of note, restitution was not accompanied by the epithelial-mesenchymal transition (EMT). EGF treatment severely affected the expression of genes important for cell-cell contact and cell communication such as selected tight junction components, CXCR4, and TFF3. Many of these genes are known to be involved in EMT and metastasis. Of special note, most of the expression changes induced by EGF are in contrast to the changes observed in migratory cells.
Cellular Physiology and Biochemistry 01/2010; 25(4-5):533-42. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rapid repair by cell migration, a process called "restitution", is essential for wound healing of mucous epithelia. Here, an established in vitro model for restitution, i.e., migration of the non-transformed intestinal epithelial cell line IEC-18 after scratch wounding, was investigated. This cell line is also known for its retained differentiation potential. The aim of this study was to test by expression profiling whether the differentiation state is altered during restitution in vitro. Using a sensitive RT-PCR method a systematic analysis of separated stationary and migratory cells was performed 48 h after in vitro wounding. Most characteristically, the differentiation state was changed in migratory cells when compared with stationary cells. For example, migratory cells lost markers of terminal differentiation and changed to a phenotype that assists the process of restitution by up-regulating the expression of genes such as plasminogen activator inhibitor-1, transforming growth factor alpha, heparin-binding EGF-like growth factor, alpha-smooth muscle actin, ornithine decarboxylase, and glyceraldehyde-3-phosphate dehydrogenase. However, there were no unequivocal signs of epithelial-mesenchymal transition (EMT) found in migratory cells.
Cellular Physiology and Biochemistry 02/2009; 24(1-2):125-32. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tff peptides are secreted mainly by the gastrointestinal epithelial cells and their primary role is maintaining normal structure and function of mucous epithelia. Ongoing studies on their expression pattern have disclosed other sites of their synthesis thus revealing additional physiological functions in the organism. Here we present new data about Tff3 expression in the cochlea of the rodent inner ear. On the basis of RT-PCR we describe the presence of Tff3 transcripts in both, a mouse cDNA library isolated from whole cochleae from postnatal days 3-15 (P3-P15), and also in cochlear tissue. By using a riboprobe for the fragment containing exon 1, 2 and 3 of Tff3, in situ hybridization, localized Tff3 signals in neurons of spiral ganglion and vestibular organ. We did not observe any abnormalities in the middle ear of Tff3 knock-out mice, neither did histological examination of the inner ear indicate any gross morphological changes in the cochlea. However, ABR (auditory evoked brain stem responses) audiograms revealed that the Tff3 knock-out animals show an accelerated presbyacusis and a hearing loss of about 15 dB at low frequencies increasing to 25 dB loss at higher frequencies. These findings suggest that Tff3 could play a role in neurosensory signaling. Further studies are needed to clarify this new function in the auditory system.
Cellular Physiology and Biochemistry 02/2008; 21(5-6):437-44. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: TFF3 (trefoil factor family 3), which is a major secretory product of the gastric antrum and the intestine, but which is nearly absent in the gastric corpus, plays a key role in the maintenance of mucosal integrity. Here, we have systematically investigated TFF3 expression in the esophagus and gastric cardia by the use of reverse transcription/polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Synthesis of TFF3, but not TFF1 or TFF2, is detectable in esophageal submucosal glands. The stratified squamous epithelium is devoid of TFF synthesis. Prominent TFF3 expression starts at the Z-line with a sharply decreasing gradient toward the cardia. Immunohistochemistry has localized TFF3 to surface mucous cells of the proximal cardia. TFF3 distribution differs characteristically from that of TFF1 (secreted primarily by superficial surface mucous cells), whereas TFF3, together with the mucin MUC5AC, is also found in deeper lying cells toward the isthmus. This is the first report of TFF3 as a typical secretory peptide of esophageal submucosal glands and gastric cardia. The different expression patterns of TFF3 and TFF1 in the cardia suggest a stepwise maturation of surface mucous cells from TFF3/MUC5AC-positive cells close to the isthmus to TFF1/TFF3/MUC5AC-positive cells at the pit. The gradient of TFF3 expression along the gastric rostro-caudal axis defines two types of gastric pit cells: those secreting TFF3 in the cardia and the antrum and those nearly devoid of TFF3 synthesis in the corpus. This indicates the special requirement, particularly of the esophagogastric junction, for TFF3-triggered protection and repair.
Cell and Tissue Research 06/2007; 328(2):365-74. · 3.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Asthma is a chronic inflammatory disease of the airways that is accompanied by goblet cell metaplasia and mucus hypersecretion. Trefoil factor family (TFF) peptides represent major secretory products of the respiratory tract and are synthesized together with mucins. In the murine lung, TFF2 is mainly expressed, whereas TFF1 transcripts represent only a minor species. TFF peptides are well known for their motogenic and anti-apoptotic effects, and they modulate the inflammatory response of bronchial epithelial cells. Here, an established mouse model of asthma was investigated (i.e., exposure to Aspergillus fumigatus [AF] antigens). RT-PCR analysis of lung tissue showed elevated levels particularly of TFF1 transcripts in AF-sensitized/challenged animals. In contrast, transcripts encoding Clara cell secretory protein (CCSP/CC10) were strongly diminished in these animals. For comparison, the expression of the goblet cell secretory granule marker mCLCA3/Gob-5, the mucins Muc1-Muc6 and Muc19, and the secretoglobins ScgB3A1 and ScgB3A2, as well as the mammalian ependymin-related gene MERP2, were monitored. Immunohistochemistry localized TFF1 mainly in cells with a mixed phenotype (e.g., TFF1-positive cells stain with the lectin wheat germ agglutinin (WGA), which recognizes mucins characteristic of goblet cells). In addition, these cells express CCSP/CC10, a Clara cell marker. When compared with mucins or CCSP/CC10, TFF1 was stored in a different population of secretory granules localized at the more basolateral portion of these cells. Thus, the results presented indicate for the first time that allergen exposure leads to the trans-differentiation of Clara cells toward a TFF1-expressing mucous phenotype.
American Journal of Respiratory Cell and Molecular Biology 04/2007; 36(3):286-95. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human airway surface epithelium is frequently damaged by inhaled factors (viruses, bacteria, xenobiotic substances) as well as by inflammatory mediators that contribute to the shedding of surface epithelial cells. To regain its protective function, the epithelium must rapidly repair and redifferentiate. The Trefoil Factor Family (TFF) peptides are secretory products of many mucous cells. TFF3, the major TFF in the airways, is able to enhance airway epithelial cell migration, but the role of this protein in differentiation has not been defined. To identify the specific role of TFF3 in the differentiation of the human airway surface epithelium, we analyzed the temporal expression pattern of TFF3, MUC5AC, and MUC5B mucins (goblet cells) and ciliated cell markers beta-tubulin (cilia) and FOXJ1 (ciliogenesis) during human airway epithelial regeneration using in vivo humanized airway xenograft and in vitro air-liquid interface (ALI) culture models. We observed that TFF3, MUC5AC, MUC5B, and ciliated cell markers were expressed in well-differentiated airway epithelium. The addition of exogenous recombinant human TFF3 to epithelial cell cultures before the initiation of differentiation resulted in no change in MUC5AC or cytokeratin 13 (CK13, basal cell marker)-positive cells, but induced an increase in the number of FOXJ1-positive cells and in the number of beta-tubulin-positive ciliated cells (P < 0.05). Furthermore, this effect on ciliated cell differentiation could be reversed by specific epidermal growth factor (EGF) receptor (EGF-R) inhibition. These results indicate that TFF3 is able to induce ciliogenesis and to promote airway epithelial ciliated cell differentiation, in part through an EGF-R-dependent pathway.
American Journal of Respiratory Cell and Molecular Biology 03/2007; 36(3):296-303. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: TFF3, a member of the TFF (trefoil factor family) peptides, and epidermal growth factor (EGF) actively support the repair of mucosal barriers, particularly during restitution. The aim of this study was to compare the motogenic effects of TFF3 and EGF.
The influence of recombinant human TFF3 (dimeric form) and EGF on the migration of IEC-18 cells was characterized in an in vitro restitution model (scratch wound assay) with the help of time-lapse video microscopy, morphometry, and immunocytochemistry including confocal laser scanning microscopy.
TFF3- and EGF-treated cells re-populated the wounded area via different migration patterns; TFF3 treatment resulted in the formation of continuous sheets of migrating cells with only a few gaps. In contrast, EGF-treated cells formed a network of migrating cells (often with a fibroblast-like morphology) with numerous gaps and only punctual contacts. TFF3 and EGF treatment also changed the localization of E-cadherin indicating endocytotic recycling and/or degradation of E-cadherin.
TFF3, in contrast to EGF, enhanced a collective cell migration ensuring a precise coverage of the re-populated area avoiding gaps.
Cellular Physiology and Biochemistry 02/2007; 20(5):329-46. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Gastrokine-2 (GKN2) is a secretory peptide of human gastric surface mucous cells (SMCs). It forms disulfide-linked heterodimers with the trefoil factor family (TFF) peptide TFF1. Binding with TFF2 was also reported. Antral SMCs differ from those of the corpus by their TFF3 expression. The aim of this study was to localize GKN2 expression along the antral gland axis, to characterize the continuous regeneration of antral glands, and to investigate the interactions of GKN2 with TFF1, TFF2 and mucins. Methods: The spatial expression of GKN1, GKN2, TFF1-3, MUC5AC and MUC6 was determined using laser microdissection and RT-PCR analysis. Furthermore, antral extracts were separated by gel chromatography and the association of GKN2 with TFF1, TFF2, and mucins was investigated. Results: Differential GKN2 expression was localized along the rostro-caudal axis of the stomach. Laser microdissection revealed characteristic differential expression profiles of GKN1, GKN2, TFF1-3, MUC5AC and MUC6 along the antral gland axis. Both GKN2 and TFF1 were expressed in superficial SMCs. Surprisingly, the TFF1-GKN2 heterodimer did not associate with the mucin fraction; whereas TFF2 showed exclusive association with mucins. Conclusions: Maturation of antral SMCs occurs stepwise via trans-differentiation of TFF3 expressing progenitor cells. The TFF1-GKN2 heterodimer and TFF2 differ characteristically by their binding to gastric mucins. This points to different physiological functions of TFF1 and TFF2, the latter maybe acting as a 'link peptide' for stabilization of the gastric mucus.
Cellular Physiology and Biochemistry 02/2007; 20(6):899-908. · 3.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rapid repair of mucous epithelia is essential for preventing inflammation which is a critical component of cancer progression.
‘Restitution’ is an early repair process which can begin within minutes and is achieved via the migration of neighbouring
cells into the wounded area. Mucosal restitution is a multistep process which requires continuous blood flow and includes
at least (i) the reduction of cell-cell contacts and a shift in the cell shape towards a migratory phenotype (characteristics
of the epithelial-mesenchymal transition), (ii) migration of cells, (iii) repolarization and formation of tight junctions
(morphological restitution) and (iv) restoration of barrier function (transmucosal epithelial resistance, functional restitution).
Secretory TFF (trefoil factor family) peptides TFF1, TFF2 and TFF3 are well known for their potent protective and healing
effects after mucosal damage (function as ‘luminal surveillance peptides’). Here, the contributions of the TFFs during the
different steps of mucosal restitution are discussed, i. e. the modulation of cell-cell contacts, their motogenic activity
and synergy with epidermal growth factor, their anti-apoptotic and pro-angiogenic effects. Special emphasis has been given
to discussion of the various signal transduction networks triggered by TFFs. It is becoming increasingly clear that these
pathways differ depending on the respective TFF.
Cellular and Molecular Life Sciences CMLS 11/2005; 62(24):2932-2938. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Injured areas of the respiratory epithelium are subject to rapid repair by the migration of adjacent epithelial cells, a process termed "restitution". Rapid re-epithelialization is promoted by interactions between migrating cells and the extracellular matrix proteins. Furthermore, epidermal growth factor (EGF) as well as trefoil factor family (TFF) peptides are well known regulators of epithelial restitution due to their motogenic effects. Migration of the human bronchial epithelial cell line BEAS-2B in modified Boyden chambers was used as a model system for airway restitution. EGF or recombinant human TFF2 or TFF3 showed mainly chemotactic activity. The motogenic response was strictly dependent upon a haptotactic substrate, but to different degrees. EGF induced phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, c-Jun-N-terminal kinase, p38, Akt, and p70S6K in BEAS-2B cells. Using specific inhibitors, the signaling cascades responsible for the motogenic response were shown to differ drastically when EGF was compared with TFF2. The motogenic effect of TFF2 was previously demonstrated to depend on ERK1/2 and protein kinase C activation; whereas the EGF-triggered motogenic response was completely independent of ERK1/2 activation but sensitive to the inhibition of phosphoinositide 3-kinase, p38, protein kinase C, or nuclear factor kappaB. However, the motogenic effects of EGF and TFF2 are additive. These data suggest that luminal EGF and TFF peptides can act synergistically in the human respiratory epithelium to enhance rapid repair processes in the course of diseases such as asthma.
American Journal of Respiratory Cell and Molecular Biology 12/2004; 31(5):528-37. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: The expression of the trefoil factor family (TFF) genes (TFF1, TFF2, and TFF3) was systematically analyzed in 18 different organs from male or female mice using RT-PCR analysis. The expression patterns showed some gender-specific differences, e.g., TFF3 transcripts in the urinary bladder and liver. Furthermore, the murine expression profile differed from that in human, e.g., in the respiratory tract and uterine cervix. As a hallmark, an aberrant TFF1-related transcript was detected specifically in the kidney and liver of several mouse strains. Molecular characterization of this rare 1.8kb long transcript from the kidney clearly revealed that its 3' region originated from the antisense strand of the TFF1 locus containing particularly large parts of the antisense strands of introns 1 and 2. Homology searches using various databases revealed that this antisense TFF1-related transcript is subject of intense alternative splicing and no protein product encoded by this antisense TFF1-related transcript could be identified. Although the function of this transcript is not known currently, we can speculate that this antisense TFF1-related transcript might have a gene silencing effect particularly on TFF1 expression in the murine kidney and liver.
[show abstract][hide abstract] ABSTRACT: At the gastric cardia, the molecular mechanisms of inflammation and metaplasia are incompletely understood. Thus, the aim of this study was to determine the expression of TFF1, TFF2 and TFF3 at this site and correlate these data with Helicobacter pylori infection or gastro-esophageal reflux disease (GERD). In 27 patients without intestinal metaplasia at the cardia, endoscopic biopsies were obtained for histology and RT-PCR. TFF1 and TFF2 were expressed in all cardia samples. TFF3 expression was significantly more frequent at the cardia (n = 15/24) than in the corpus (n = 2/26). TFF3 expression at the cardia was mainly observed in GERD patients, and there was a clear tendency towards higher interleukin-8 (IL-8) transcription levels; whereas TFF3 expression was not correlated with the H. pylori status or to tumor necrosis factor-alpha (TNF-alpha) expression. The expression of TFF3 at the cardia may represent an adaptation to GERD and precede the development of Barrett's esophagus.
[show abstract][hide abstract] ABSTRACT: The objective of this study was to determine whether the lacrimal gland synthesizes mucins and whether they are changed with age or in cases of dry eye. Expression of mucins in human lacrimal glands was monitored by reverse transcription-polymerase chain reaction analysis. Furthermore, the presence and distribution of MUC1, -2, -4, -5AC, -5B, -6 and -7 in epithelia of the human lacrimal gland and its excretory duct system were assessed with antisera to mucin peptide cores. Thirty normal tissues from cadavers of different ages were tested, plus four with dry eye treated with artificial tears. Expression studies detected mRNAs for mucins MUC1, -4, -5AC, -5B, -6 and -7; whereas the MUC2 message was absent. The message for MUC4 was present in all four cases of dry eye, but only in six out of the 30 normal glands from individuals who did not receive artificial tears. MUC6 mRNA was detected only in about half of the investigated samples. Immunohistochemistry revealed membrane-bound MUC1 at the apical surface of acinar cells, absence of MUC2, MUC5AC associated with goblet cells of excretory ducts, MUC5B and -7 in the cytoplasm of acinar cells, and MUC7 also in epithelial cells of excretory ducts. MUC4 mucin was detected only in those individuals in which message was identified. In dry eyes, MUC5AC and -5B were localized in the same acinar cells; whereas MUC2 and MUC6 were not detectable. Dot-blot analysis clearly revealed increased amounts of MUC4, -5AC, and -5B in the glands of elderly women who received treatment for dry eyes. These results confirm that the human lacrimal gland synthesizes a spectrum of mucins; part of them might be correlated with age.
Cell and Tissue Research 06/2004; 316(2):167-77. · 3.68 Impact Factor